Bis(pivaloyloxymethyl) thymidine 5'-phosphate is a cell membrane-permeable precursor of thymidine 5'-phosphate in thymidine kinase deficient CCRF CEM cells

Bis(pivaloyloxymethyl) thymidine 5-phosphate (POM(2)-dTMP) has been investigated as a membrane-permeable prodrugs of dTMP. The growth inhibitory activity of POM(2)-TMP has been compared with thymidine (TdR) in wild type CCRF CEM cells (CEM) and a strain that lacks TdR kinase (CEM tk-). After 72 h incubation at 37 degrees C, TdR showed significant antiproliferative activity (IC(50)=27 microM) against CEM cells but was weakly effective (IC(50)=730 microM) against the mutant cell line. By comparison, bis(pivaloyloxymethyl) thymidine 5'-monophosphate (POM(2)-dTMP) was equally inhibitory (IC(50)=5 microM) to both cell lines. The growth inhibitory effects were reversed by deoxycytidine. Cellular [methyl-(3)H]dTTP pools increased linearly over 2h during incubation of CEM or CEM tk- with 5 microM POM(2)-[methyl-(3)H]dTMP. The incorporation of [methyl-(3)H]TdR into HClO(4)-insoluble cell residue by CEM tk- was <0.1% that of CEM and did not increase over 1h. In contrast, CEM tk- incorporated radioactivity from POM(2)-dTMP into acid insoluble residue at a rate 59% that of CEM. These results demonstrate that POM(2)-dTMP can penetrate into cells and serve as a source of dTMP.     

Genesis of the primate neostriatum: [3H]thymidine autoradiographic analysis of the time of neuron origin in the rhesus monkey.

The time of neuron origin and the spatio-temporal pattern of neuronal distribution in the caudate nucleus and putamen were examined in autoradiograms from 2- to 5- month-old monkeys that were exposed to a single pulse of [³H]thymidine on various embryonic or early postnatal days. Heavily labeled neurons, representing cells that entered their last cell division at the time of [³H]thymidine injection, were found in both the caudate and putamen in the animals injected between embryonic days 36 and 80, but not in monkeys injected at earlier or later stages. A peak in neuronal proliferation occurs between embryonic days 43 and 50. Thus, all of the neurons that ultimately compose the neostriatum are generated during a 45-day period in the first half of gestation, which lasts 165 days in the Rhesus monkey.Two classes of neurons, distinguished by their size and morphology, were discernible in autoradiograms stained with toluidine blue: small neurons with maximum cell body diameters ranging from 13 to 22 μm and larger cells with maximum diameters of 25–40 μm. The larger cells are generated over the first 7–8 days of striatal neurogenesis, while small cells are produced throughout the entire period. In agreement with previous [³H]thymidine autoradiographic studies in rodents, no spatio-temporal gradient related to the time of neuron origin was found in the primate neostriatum. However, small neurons generated on a given day were usually arranged in scattered clusters which, in a single coronal section, consisted of 5 to over 40 labeled neurons. These isochronously generated cell aggregates may represent a basic structural unit of the neostriatum.     

肝癌患者CIK细胞的诱导及对肝癌细胞毒作用的研究

观察肝癌患者PBMC在体外诱导成CIK细胞的能力及其对肝癌细胞的细胞毒作用。对比正常人组和肝癌患者组CIK细胞和LAK细胞之间的扩增差异。用流式细胞仪检测CIK细胞表面标志CD3、CD5 6 ,3 H TdR释放法检测CIK细胞、LAK细胞对肝癌细胞系SMMC 772 1等多种细胞系的细胞毒作用。结果显示 ,肝癌组和正常组的CIK细胞扩增倍数分别达 6 4 3倍和6 7 4倍 ,CD3+CD5 6 +细胞扩增倍数均达 6 0 0倍以上 ,在细胞扩增曲线及细胞表面标志上无差异 ,远大于LAK细胞 ;肝癌组CIK对肝癌细胞SMMC 772 1、Bel 74 0 2、Hep 3b杀伤能力均达 6 5 %～ 81% ,与正常组相同 ,且与对肠癌细胞系HIC 2 5 1杀伤能力无差异 ;对正常胎肝细胞系L 0 2的细胞毒作用 <5 % ;肝癌患者CIK对耐药的和未诱导耐药的K5 6 2细胞细胞毒作用均达到 70 %左右。肝癌患者CIK和正常人CIK一样对肝癌细胞有很强的细胞毒作用 ,对耐药肿瘤同样有效 ,对正常肝细胞无损伤 ,具有临床应用前景     

细粒棘球蚴病患者细胞免疫应答功能的测定和分析

用单克隆抗体免疫酶标染色法,分析40例棘球蚴病人外周血T淋巴细胞及其亚群的变化。并用PHA刺激(~3H)TdR掺入法测定病人外周血T淋巴细胞转化功能及对外周血淋巴细胞经ConA诱生的白细胞介素2(IL-2)的活性进行测定,以评价棘球蚴病人细胞免疫应答反应水平。结果:病人外周血总T淋巴细胞百分比正常,T辅助细胞(OKT_4~+)百分比降低,T抑制细胞(OKT_8~+)百分比增高,OKT+4~+/OKT_8~+比值下降(1.06±0.07,正常人1.70±0.11,P<0.01),Tac~+细胞百分比增高(8.62±0.81,正常人2.90±0.61,P<0.01),T淋巴细胞转化功能增高(242.49±16.68),正常人116.27+26.01,P<0.01),但外周血IL-2活性与正常人无显著差异。提示棘球蚴病人有T淋巴细胞亚群水平的免疫抑制。     

Proliferating cell nuclear antigen (PCNA) expression in regenerating rat liver after partial hepatectomy

Proliferating cell nuclear antigen (PCNA) is a nuclear protein maximally elevated in the S phase of proliferating and transformed cells and is recognized by the monoclonal antibody PC-10 in paraffin tissue sections. The liver regenerative process after partial hepatectomy in rats was estimated with thein vivo incorporation of [³H]thymidine into liver DNA and the liver thymidine kinase activity. The expression of PCNA in rat liver after partial hepatectomy was performed by immunohistochemical staining with PC-10 in paraffin embedded tissues, at different time intervals up to 240 hr. Proliferating cell nuclear antigen expression, [³H]thymidine incorporation into DNA, and liver thymidine kinase activity exhibited marked oscillations during the liver regenerative process. A close relationship was demonstrated among DNA synthesis, thymidine kinase activity, and PC-10 score. Our results suggest that PC-10 monoclonal antibody may be used as a worthwhile proliferation index in the evaluation of the rate of liver regeneration in rats.     

in vitro metabolism of 1-β-d-arabinofuranosylcytosine and 1-β-2'-fluoro-arabino-5-iodocytosine in normal and herpes simplex type 1 virus-infected cells

Phosphorylation of 1-β-D-2'-F-arabino-5-iodocytosine (FIAC), a newly synthesized pyrimidine nucleoside with potent antiherpesvirus activity, was compared with that of its parent compound, 1-β-D-arabinofuranosylcytosine (ara-C). While ara-C was phosphorylated extensively by homogenates of normal, rapidly proliferating mouse tissues, FIAC was a poor substrate for the nucleoside kinase occurring in such normal tissues. With cell homogenates of noninfected Vero cells, thymidine (TdR) was phosphorylated about fifty and twenty times more efficiently than FIAC and ara-C, while infection of Vero cells with Herpes Simplex Virus Type 1 (HSV-1) resulted in a 23-fold increase of TdR- and a 1270-fold increase of FIAC phosphorylation. In contrast, phosphorylation of ara-C was increased only by a factor of 2.6. While the reaction products obtained with homogenates of normal mouse tissues were 5'-mono-, di- and triphosphates of ara-C and FIAC, the reaction products with noninfected and infected Vero cell homogenates were predominantly monophosphates. In contrast, TdR was efficiently phosphorylated to its 5'-mono-, di- and triphosphates by such homogenates. In intact HSV-1-infected Vero cells, FIAC was rapidly taken up and phosphorylated to FIACMP and to an as yet unidentified metabolite. In contrast, TdR was taken up and phosphorylated to 5'-mono-, di- and triphosphates and ara-C was taken up moderately but metabolized poorly to its 5'-mono-, di- and triphosphates. Thus, in normal tissues, FIAC was a poorer substrate than ara-C for nucleoside kinases, but in intact HSV-1-infected Vero cells FIAC was efficiently phosphorylated and thus behaved like a TdR analog, except that it was phosphorylated only to the 5'-monophosphate and a hitherto unidentified metabolite. The greatly increased phosphorylation of FIAC by HSV-1-infected Vero cells probably accounts, at least in part, for its great selectivity of action.     

Adenosine stimulation of the proliferation of colorectal carcinoma cell lines. Roles of cell density and adenosine metabolism

Adenosine is a purine nucleoside which is present at micromolar concentrations in the extracellular fluid of solid cancers as a result of tissue hypoxia. Adenosine acts to promote tumor survival by inhibiting the cell-mediated anti-tumor immune response. However, its role in modulating proliferation of the tumor cell population is unclear. Differing results have been obtained using adenosine analogues or by interfering with adenosine metabolism. We examined the effect of adenosine itself on DNA synthesis and cell growth in six different human and mouse colorectal carcinoma cell lines, from different sites and at different stages of differentiation. Adenosine given as a single dose consistently stimulated DNA synthesis and cell proliferation in all cell lines tested, with an EC(50) of 3.8-30 microM and a maximum stimulation being reached at 10-100 microM. AMP and ATP also stimulated cell proliferation at similar doses. The stimulation by adenosine varied depending upon the culture cell density, with the greatest mitogenic effect at subconfluent densities. Adenosine was metabolized by cellular adenosine deaminase and adenosine kinase. The half-life (t(1/2)) for the decline in adenosine concentration in the medium following a single addition was between 40 min and 3 hr depending on the cell line and culture conditions. The rate of production of endogenous adenosine was low under normoxic culture conditions. Continuous dosing of cultures with adenosine to provide a steady-state concentration showed that proliferation could be stimulated by low micromolar concentrations of adenosine. We conclude that adenosine is stimulatory to the growth of human colorectal carcinoma cells at concentrations present within the tumor extracellular environment.     

Induction of benzpyrene hydroxylase in fetal liver explants by flavones and phenobarbital

The addition of β-naphthoflavone (BNP) to fetal rat liver explants was accompanied by an increase in benzpyrene (BP) hydroxylase activity. The concentration of BNF which produced a one-half maximal induction was approximately 3 × 10^?7 M. (The term “induction” is used in a general sense to describe an elevation in enzyme activity, without specifying a genetic mechanism.) A lag of induction of 12 hr was observed when BNF was added to fresh cultures. This lag of induction was mitigated when BNF was added to 22- and 44-hr preincubated explants. Maximal induction was observed at 48–74 hr after the addition of BNF at 10^?5 M to 44-hr preincubated cultures. The early but not the later phase of BNF-mediated induction of BP hydroxylase was blocked by mercapto-(pyridethyl)-benzimidazole, an inhibitor of RNA synthesis, while both phases of induction were blocked by cycloheximide, an inhibitor of protein synthesis. The results are in accord with the hypothesis that BNF increases the de novo synthesis of BP hydroxylase in the fetal rat liver explant system. Pretreatment of fetal liver explants with pentamethoxyflavone (PMF) resulted in a decreased induction of BP hydroxylase by either BNF or 4'-bromoflavone (BrF), the latter being a much more potent inducer in this system. Since it was recently reported that the flavone derivative, PMF, was unable to induce BP hydroxylase in this system over a wide dose range, the results suggest that a common receptor interaction is required prior to the induction of BP hydroxylase by flavones.     

环孢霉素A对体外培养人视网膜色素上皮细胞的抑制

为寻找抑制玻璃体内细胞增生的有效药物,用³H-胸腺嗜啶核苷(²H—TdR)掺入及液体闪烁技术,观察了环孢霉素A(CsA)(0.125"μg/ml~4.0μg/ml)对体外培养的加或不加巨噬细胞培养调理液(MCM)的人视网膜色素上皮(RPE)细胞增生的影响。CsA在0.125μg/ml时细胞的抑制率为-3.8%~4.1%(P>0.05);在0.25μg/ml～4.0μg/ml时为8.4%~95.1%(P<0.05),且呈剂量依赖性(r＝0.9413)(P<0.001),ID₅₀为1.49μg/ml。加MCM组细胞增殖较对照组明显（3d,26%;5d,34%;P<0.05),CsA对细胞增殖抑制率较对照组有提高(1.0%～13.9%)．表明CsA对体外培养的人RPE细胞有明显的抑制作用． （中华眼底病杂志,1995,11：22-24）